Method for treating neoplasms using hollow silica spheres

ABSTRACT

The invention pertains to a method for treating a neoplasm, such as colorectal cancer, using hollow silica spheres (“HSS”). It also is directed to a method for making uncalcined HSS, calcined HSS from which phenyl groups have been removed, and HSS incorporating particles of Fe3O4, as well as compositions containing HSS

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to U.S. application Ser. No. 15/995,904,filed Jun. 1, 2018, which is hereby incorporated by reference for allpurposes.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR

Related aspects of this technology are described by Akhtar, et al., J.Saudi Chemical Society (available online Sep. 22, 2018), which isincorporated by reference for all purposes.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of medicine and biopolymerchemistry, especially to use of hollow silica spheres (“HSS”) fortreatment of neoplasms such as colorectal cancer.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentdisclosure.

Silica nanoparticles are used in many fields. They are used as scaffoldsor carriers for chemical and biological materials, such as drugs orcatalysts and also have been incorporated into lithium batteries,ceramics and sensors; Uysal, B. O., Tepehan, F. Z. Controlling thegrowth of particle size and size distribution of silica nanoparticles bythe thin film structure. J. Sol-Gel Sci. Technol., 63 (1) (2012), pp.177-18.

Silica nanoparticles are used to enhance the mechanical capabilities andincrease the strength of thin films including anticorrosion films andsuperhydrophobic films; Ramezani, M., M. R. Vaezi, A. Kazemzadeh,Preparation of silane-functionalized silica films via two-step dipcoating sol-gel and evaluation of their superhydrophobic properties.Appl. Surf. Sci., 317 (2014), pp. 147-153; Akhtar, S., et al.Enhancement of anticorrosion property of 304 stainless steel usingsilane coatings. Appl. Surf. Sci., 440 (2018), pp. 1286-1297.

Hollow silica spheres (HSS) having dimensions in the nanometer tomicrometer ranges have emerged as a versatile material for manyindustrial, medical and scientific applications, including boththerapeutic and diagnostic application. These include their use in drugdelivery or biomolecular release systems and as imaging agents formedical diagnostics; Adhikari, C., et al. Drug delivery system composedof mesoporous silica and hollow mesoporous silica nanospheres forchemotherapeutic drug delivery. J. Drug Delivery Sci. Technol., 45(2018), pp. 303-314; Rosu, C., et al. Polypeptide-coated silicaparticles dispersed in lyotropic liquid crystals of the samepolypeptide. J. Phys. Chem. B, 120 (29) (2016), pp. 7275-7288; Jiang,Y., D. Mu, S. Chen, B. Wu, Z. Zhao, Y. Wu, Z. Ding, F. Wu. Hollow silicaspheres with facile carbon modification as an anode material forlithium-ion batteries. J. Alloy Compd., 744 (2018), pp. 7-14; Jang J Y,Duong H T T, Lee S M, Kim H J, Ko Y J, Jeong J H, Lee D S, Thambi T, SonS U. Folate decorated hollow spheres of microporous organic networks asdrug delivery materials. Chem Commun (Camb). 2018 Apr. 5;54(29):3652-3655. doi: 10.1039/c8cc01240g; Yang Y, Lu Y, Abbaraju P L,Zhang J, Zhang M, Xiang G, Yu C. Multi-shelled Dendritic MesoporousOrganosilica Hollow Spheres: Roles of Composition and Architecture inCancer Immunotherapy. Angew Chem Int Ed Engl. 2017 Jul. 10;56(29):8446-8450. doi: 10.1002/anie.201701550. Epub 2017 May 3; Lv R,Zhong C, Gulzar A, Gai S, He F, Gu R, Zhang S, Yang G, Yang P.Synthesis, luminescence, and anti-tumor properties ofMgSiO₃:Eu-DOX-DPP-RGD hollow microspheres. Dalton Trans. 2015 Nov. 14;44(42):18585-95. doi: 10.1039/c5dt03604f. Epub 2015 Oct. 8; Yang D, YangG, Wang X, Lv R, Gai S, He F, Gulzar A, Yang P. Y2O3:Yb,Er@mSiO₂—Cu(x)Sdouble-shelled hollow spheres for enhancedchemo-/photothermal-anti-cancer therapy and dual-modal imaging.Nanoscale. 2015 Jul. 28; 7(28):12180-91. doi: 10.1039/c5nr02269j. Epub2015 Jul. 1; Teng Z, Su X, Zheng Y, Zhang J, Liu Y, Wang S, Wu J, ChenG, Wang J, Zhao D, Lu G. A Facile Multi-interface TransformationApproach to Monodisperse Multiple-Shelled Periodic MesoporousOrganosilica Hollow Spheres. J Am Chem Soc. 2015 Jun. 24;137(24):7935-44. doi: 10.1021/jacs.5b05369. Epub 2015 Jun. 11; She X,Chen L, Velleman L, Li C, Zhu H, He C, Wang T, Shigdar S, Duan W, KongL. Fabrication of high specificity hollow mesoporous silicananoparticles assisted by Eudragit for targeted drug delivery. J ColloidInterface Sci. 2015 May 1; 445:151-160. doi: 10.1016/j.jcis.2014.12.053.Epub 2014 Dec. 25; Mohapatra S, Rout S R, Narayan R, Maiti T K.Multifunctional mesoporous hollow silica nanocapsules for targetedco-delivery of cisplatin-pemetrexed and MR imaging. Dalton Trans. 2014Nov. 14; 43(42):15841-50. doi: 10.1039/c4dt02144d; Chang F P, Hung Y,Chang J H, Lin C H, Mou C Y. Enzyme encapsulated hollow silicananospheres for intracellular biocatalysis. ACS Appl Mater Interfaces.2014 May 14; 6(9):6883-90. doi: 10.1021/am500701c. Epub 2014 Apr. 15;Chen Y, Chen H R, Shi J L. Construction of homogenous/heterogeneoushollow mesoporous silica nanostructures by silica-etching chemistry:principles, synthesis, and applications. Acc Chem Res. 2014 Jan. 21;47(1):125-37. doi: 10.1021/ar400091e. Epub 2013 Aug. 14.

Despite the many benefits of hollow silica spheres they are have notbeen successful as agents for cancer therapy. The inventors believe thatthis is accounted for by the presence of phenyl groups which may impairor prevent the HSS from exhibiting pharmacokinetic and pharmacodynamicsproperties useful for treating cancer and other neoplasms. HSS bearingphenyl groups are resistant to degradation in the body due to theinability or relatively poor ability (compared to HSS without phenylgroups) of the body to metabolize phenyl groups through the liver andbile and thus be easily cleared from the body. HSS that remain in thebody pose significant safety and health hazards, for example due to thepresence of the phenyl groups which may form be released as benzene.

The inventors have overcome the problem associated with the presence ofphenyl groups and show that HSS without phenyl groups and/or HSS whichincorporate Fe₃O₄ particles, surprisingly exhibit superior anti-cancerproperties. Accordingly, it is one object of the present invention toprovide a method to synthesize HSS and efficiently remove non-degradablephenyl groups and to produce hollow silica spheres containing Fe₃O₄ andthe hollow nanoparticles obtained therefore. Unlike many prior processesof making HSS, this method does not require the use of template.

BRIEF SUMMARY OF THE INVENTION

The invention pertains to a method for treating a neoplasm, such ascolorectal cancer, using hollow silica spheres (“HSS”). It also isdirected to a method for making uncalcined HSS, calcined HSS from whichphenyl groups have been removed, and HSS incorporating particles ofFe₃O₄, as well as compositions containing HSS.

The following non-limited embodiments discloses particular aspects ofthis technology.

According to a first aspect of the invention, the present disclosurerelates to a method for treating a subject having a neoplasm comprisingcontacting a neoplastic cell with hollow silica spheres (“HSS”) selectedfrom the group consisting of at least one of an uncalcined hollow silicasphere (“u-HSS”), a calcined hollow silica sphere (“c-HSS”) or aFe₃O₄-hollow silica sphere (“Fe—HSS”), wherein said hollow silicaspheres are substantially free of phenyl groups.

In some embodiments of this method, the hollow silica spheres areprepared by a process comprising hydrolysis of phenyl-tri-methoxysilane(“PTMS”) or another hydrolyzable aryl silane followed by condensationwith a hydroxide base.

In some embodiments of the invention the HSS are u-HSS, c-HSS and/orFe—HSS.

In some embodiments the HSS further comprise at least one anticancerdrug or agent.

In some embodiments, the neoplasm is cancer.

In some embodiments, the neoplasm is colon or colorectal cancer.

In some embodiments, the neoplasm is at least one of non-melanoma skincancer, breast cancer, lung cancer, prostate cancer, melanoma, bladdercancer, non-Hodgkin's lymphoma, kidney cancer, leukemia, pancreaticcancer, thyroid cancer, liver cancer, endometrial cancer, throat cancer,ovarian cancer, or testicular cancer.

In some embodiments the neoplasm is a benign neoplasm.

In some embodiments the neoplasm is a pre-cancerous tumor orprecancerous lesion.

In some embodiments, the HSS are administered in situ to the site of atumor or cancer cells.

In some embodiments, the HSS are administered parenterally.

In some embodiments, the HSS are administered subcutaneously,intramuscularly, or intravenously.

In some embodiments, the HSS are administered tropically.

In some embodiments, the HSS are administered orally.

In some embodiments an amount of HSS is administered sufficient todisrupt the nuclear membrane of a neoplastic cell for example byinducing nuclear condensation, augmentation, and/ordisintegration/fragmentation. In other embodiments, an amount of HSS isadministered sufficient to disrupt the cellular membrane of a cancercell.

Another aspect of the invention is directed to a composition comprisinghollow silica spheres (“HSS”) selected from the group consisting of atleast one of an uncalcined hollow silica sphere (“u-HSS”), a calcinedhollow silica sphere (“c-HSS”) or a Fe₃O₄-hollow silica sphere(“Fe—HSS”). Preferably, the hollow silica spheres are substantially freeof phenyl or other aryl groups, for example, c-HSS may contain <0.01,0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, or 50 molar % of the phenylor aryl groups present on corresponding uncalcined HSS such as the u-HSSdisclosed herein. All phenyl groups on the surface and in the core canbe removed via calcination and this can be confirmed by TGA and FT-IR.For example, uncalcined Fe—HSS may be calcined to remove all or aportion of the phenyl groups and/or to reduce the size of the calcinedFe—HSS compared to uncalcined Fe—HSS. Calcined HSS are typically smallerthat the corresponding u-HSS, for example, they may be 5, 10, 15, 20,25, 30, 35, 40, 45 or 50% smaller in average diameter than correspondingu-HSS.

In some embodiments, the u-HSS have an average diameter ranging from660, 710, 760, 810, to 860 nm or any intermediate value within thisrange.

In some embodiments the c-HSS and Fe—HSS have average diameters rangingfrom 415, 465, 515, 565 to 615 nm or any intermediate value within thisrange.

In some embodiments Fe—HSS will contain about <0.05, 0.05, 0.1, 0.2,0.5, 1, 2, 5, 10, to >10 wt % Fe₃O₄ particles based on the total weightof the Fe—HSS (e.g., the weight of the Fe₃O₄ particles+the weight of theHSS). These particles may consist of, consist essentially of, orcomprise Fe₃O₄. In some embodiments, other types of magnetic materialsmay be substituted in whole or part for Fe₃O₄, these other materialsinclude corresponding Ni or Co compounds such as NiO or Co₃O₄ based ontotal weight of the Fe—HSS. In other embodiments eithersuperparamagnetic iron oxide particles with a mean hydrodynamic diameterof >50 nm such as feruxomides or Feridex IV may be used. In still otherembodiments ultra small superparamagnetic iron oxide particles with ahydrodynamic diameter of <50 nm such as Ferumoxtran-10 may beincorporated into HSS. In some embodiments, the silica-containing shellof the HSS is thicker than its core and in other embodiments, the coreis thicker than the shell.

In some embodiments, the silica-containing shell has a thickness ofabout 150, 160, 170, 180, 190, 200 to 210 nm or any intermediate valuewithin this range, and the core has a diameter of about 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220 to 230 nm or anyintermediate value within this range.

In some embodiments the HSS is Fe—HSS that comprises Fe₃O₄ particleshaving an average diameter ranging from 9.5, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21 to 21.3 nm or any intermediate value within thisrange.

In some embodiments, the hollow silica spheres are monodisperse with acoefficient of variation, defined as a ratio of the standard deviationto the mean diameter of the hollow silica spheres, of less than 1, 2, 3,4 or 5%.

In some embodiments, the hollow silica spheres have a solubility inwater of 0.1, 0.2, 0.5, 1, 1.2, 1.5, 2, 5, 10, 20, to 50 mg per 10 mL ofwater or any intermediate value within this range.

In some embodiments, the hollow silica spheres have a specific surfacearea of 350, 360, 370, 380, 390, 400, 410, 420, 430, 440 to 450 m²/g orany intermediate value within this range.

In some embodiments, the hollow silica spheres have a t-plot externalsurface area of 40, 45, 50, 55, 60, 65, 670 to 75 m²/g or anyintermediate value within this range.

In some embodiments, the hollow silica spheres have an average porediameter of 1.7 to 8 nm with a cumulative pore volume of 0.02, 0.025,0.03, to 0.035 cm³/g or any intermediate value within this range.

In some embodiments, the HSS further comprise at least one anticancerdrug or agent.

In some embodiments, the HSS further comprise a coating such as acationic polymer such as chitosan.

In some embodiments, the HSS further comprise a targeting agent, such asa tumor-specific antibody or antibody fragment such as Fab or Fab2 oranother ligand that binds to neoplastic, cancer or tumor cells.

Another aspect of the invention sis directed to a method for forminghollow silica spheres (“HSS”) comprising: dissolving a hydrolyzable arylsilane in an aqueous solution comprising water and an acid to form ahydrolyzed silane solution; mixing the hydrolyzed silane solution with ahydroxide base to form a precipitate; and recovering hollow silicaspheres from the precipitate.

In some embodiments of this method the hydrolyzable aryl silane istrimethoxy(phenyl) silane.

In some embodiments of this method the hydroxide base is NH₄OH.

In some embodiments, this method further comprises calcining the silicaspheres at a temperature that removes phenyl groups from uncalcined HSS,thereby forming c-HSS.

In some embodiments, the method further comprises incorporating Fe₃O₄into the aqueous solution containing the hydrolyzable aryl silane, andadding the hydroxide base, thereby forming Fe—HSS.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings below.

FIGS. 1A-1C: Substrates and sol-gel process (FIG. 1A) used to prepareFe₃O₄ nanoparticles incorporated hollow silica spheres or Fe—HSS (FIG.1B). A photo of the prepared product is shown in FIG. 1C.

FIGS. 2A-2F. Morphology and size analysis of the Fe₃O₄ nanoparticles andhollow silica spheres (HSS). (FIG. 2A) TEM image and (FIG. 2B) sizehistogram of Fe₃O₄ particles. The average diameter of the particles is˜14 nm. (FIG. 2C) SAED pattern of the Fe₃O₄ nanoparticles, showing thepolycrystalline nature, the first 5-rings of the pattern started fromthe inner ring are indexed as, (220), (311), (400), (511) and (440). SEMmicrograph of (FIG. 2D) u-HSS, (FIG. 2E) c-HSS and (FIG. 2F) Fe—HSS.

FIGS. 3A-3D. Morphology and size analysis of hollow silica spheres(HSS). TEM image of (FIG. 3A) u-HSS, (FIG. 3B) c-HSS and (FIGS. 3C and3D) Fe—HSS. A shell and a core are marked in FIG. 3B. The Fe₃O₄nanoparticles inside the HSS are highlighted by white arrows in FIG. 3D.

FIG. 4. XRD pattern of Fe—HSS specimen. The main peaks of Fe₃O₄ areindexed in the pattern. The broad peak marked with * is related toamorphous silica.

FIGS. 5A-5D. A digital photograph of physical appearance of (FIG. 5A)c-HSS and (FIG. 5B). A digital photograph of the magnetic slab toattract the Fe₃O₄ powder (FIG. 5C) before and (FIG. 5D) after performingthe test. The Fe—HSS powder can be seen attached to the magnetic slab.

FIG. 6. TGA plots of the u-HSS (a) orange plot, c-HSS (a) red plot, andFe—HSS (c) blue plot.

FIGS. 7A-7E. Morphology of cancer cells morphology after treatment ofUncal-HSS: The HCT-116 cells analyzed 48 hrs post-treatments. FIG.sshowed the effects of HSS-1, (FIG. 7A) control, (FIG. 7B) treated withdose 2 ul/0.1 ml, (FIG. 7C) treated with 4 ul/0.1 ml, (FIG. 7D) treatedwith 6 ul/0.1 ml and (FIG. 7E) treated with 8 ul/0.1 mL. Arrows showcellular disintegration of cancer cells. 400× magnifications

FIGS. 8A-8E. Morphology of cancer cells morphology after treatment ofCal-HSS: The HCT-116 cells analyzed 48 hrs post-treatments. FIG.s showedthe effects of HSS-2, (FIG. 8A) control, (FIG. 8B) treated with dose 2ul/0.1 ml, (FIG. 8C) treated with 4 ul/0.1 ml, (FIG. 8D) treated with 6ul/0.1 ml and (FIG. 8E) treated with 8 ul/0.1 mL. Arrows show cellulardisintegration of cancer cells. 400× magnifications

FIGS. 9A-9E. Morphology of cancer cells morphology after treatment ofFe—HSS: The HCT-116 cells analyzed 48 hrs post-treatments. FIGS. showedthe effects of HSS-3, (FIG. 9A) control, (FIG. 9B) treated with dose 2ul/0.1 ml, (FIG. 9C) treated with 4 ul/0.1 ml, (FIG. 9D) treated with 6ul/0.1 ml and (FIG. 9E) treated with 8 ul/0.1 mL. Arrows show cancercell nuclear augmentation and condensation. FIGS. 9D and 9E showcomplete disintegration of cell bodies. 400× magnifications.

FIG. 10. Cancer cell viability by MTT Assay. The HCT-116 cells weretreated with Uncal-HSS (1 mg/mL, 3 mg/mL, 5 mg/mL, 7 mg/mL) for 48 hrs.Data are the means±SD of three different experiments. Difference betweentwo treatment groups were analysed by student's t test where (*p<0.05),p-values were calculated by Student's t-test. No changes were observedin control group.

FIG. 11. Cancer cell viability by MTT Assay. The HCT-116 cells weretreated with Cal-HSS (1 mg/mL, 3 mg/mL, 5 mg/mL, 7 mg/mL) for 48 hrs.Data are the means±SD of three different experiments. Difference betweentwo treatment groups were analyzed by student's t test where (*p<0.05,**p<0.01), p-values were calculated by Student's t-test. No changes wereobserved in control group.

FIG. 12. Cancer cell viability by MTT Assay. The HCT-116 cells weretreated with Fe—HSS (1 mg/mL, 3 mg/mL, 5 mg/mL, 7 mg/mL) for 48 hrs.Data show the means±SD of three different experiments. Differencesbetween two treatment groups were analyzed by student's t test where(*p<0.05, **p<0.01; ***p<0.001), p-values were calculated by Student'st-test. No changes were observed in control group.

DETAILED DESCRIPTION OF THE INVENTION

As explained above the inventors have now synthesized and compareduncalcined-hollow silica spheres (u-HSS), calcined hollow silica spheres(c-HSS) and Fe₃O₄-hollow silica spheres (Fe—HSS). Calcination was usedto remove phenyl groups. Further detail about the substrates for HSS andthe steps of producing and analyzing anti-cancer properties of HSS areprovided below.

Silica Spheres.

When referencing hollow silica spheres, “hollow” refers to a centralarea (i.e., a core portion) of a particle which has a lower density ofsilica compared to the surrounding structure (i.e., the shell portion).While the definition of “hollow” may encompass a continuous void that iscompletely free of silica, this is not a requirement, and some silicamay be disposed within the core portion. By way of example, a silicaparticle which has a substantially continuous density of silica from onepoint on the particle though the center of the particle to a pointdirectly across from it would be considered solid herein and not hollow,whereas a silica particle that has 60-80 wt. % of a total silica contentlocated in the shell portion, with the remaining 20-40 wt. % of a totalsilica content located in the central area would be considered hollowherein.

The “degree of hollowness” of the hollow silica spheres as used hereinis an indicator of the density differential between thesilica-containing shell and the core, with higher degrees of hollownessbeing associated with an increased capacity for storage (e.g., ofpharmaceutical or cosmetic payloads), adsorption, etc. The degree ofhollowness is defined as a maximum peak intensity of the core divided bya minimum peak intensity of the silica-containing shell, each of whichare measured by transmission electron microscopy. That is, given thehigher density of silica in the silica-containing shell than in thecore, it is more difficult for a beam of electrons to pass through thesilica-containing shell, resulting in intensity profiles that can beused to quantify this silica density disparity. The degrees ofhollowness can then be calculated for the individual hollow silicaspheres and averaged. In some embodiments, the hollow silica spheresproduced herein have an average degree of hollowness of 3, 4, 5, 6, 7 to8, preferably 3.2 to 7.5, preferably 3.4 to 7.0, preferably 3.6 to 6.5,preferably 3.8 to 6.0, preferably 4.0 to 5.5, preferably about 4.06.Such a degree in hollowness is much higher (i.e., more hollow) thansilica spheres which have not been calcined, which have an averagedegree of hollowness of about 2.3.

HSS Shape.

The shape of the core may generally determine the shape of the hollowsilica spheres. In a preferred embodiment, the hollow silica spheres arespherical or substantially spherical. Sphericity is a measure of howclosely the shape approaches that of a mathematically perfect sphere,and is defined as the ratio of the surface area of a perfect spherehaving the same volume as a hollow silica sphere to the surface area ofthe hollow silica sphere (with unity being a perfect sphere). Preferablythe hollow silica spheres have a high sphericity, with an averagesphericity of at least 0.9, preferably at least 0.92, preferably atleast 0.94, preferably at least 0.96, preferably at least 0.98,preferably at least 0.99. In some embodiments, the hollow silica spheresare classified based on roundness, and are categorized herein as beingsub-rounded, rounded, or well-rounded, preferably well-rounded, usingvisual inspection similar to characterization used in the Shepard andYoung comparison chart.

It is also envisaged that hollow silica particles may be manufactured inshapes other than spheres having high sphericities and roundness asdescribed above. By way of example, particles may be produced in shapessuch as rods, cylinders, rectangles, triangles, pentagons, hexagons,prisms, disks, platelets, cubes, cuboids, flakes, stars, flowers, andurchins (e.g. a globular particle possessing a spiky uneven surface).

In preferred embodiments, the methods disclosed herein produce hollowsilica spheres which are uniform. As used herein, the term “uniform”refers to no more than 10%, preferably no more than 5%, preferably nomore than 4%, preferably no more than 3%, preferably no more than 2%,preferably no more than 1% of the distribution of the hollow silicaspheres having a different shape. For example, the hollow silica spheresare highly spherical (e.g., have an average sphericity of at least 0.9)and have no more than 1% of nanocomposite hollow particles in an oblongshape. Included in this definition of “uniform” is the degree in whichthe hollow silica spheres remain intact. In preferred embodiments, thesilica-containing shell completely surrounds the hollow core, so that nofluid or compound may ingress into or egress out of the core exceptthrough pores located within the silica-containing shell. However, whena sphere is ruptured slightly so that silica-containing shell does notcompletely surround the core, the ruptured sphere tends to take on anappearance of a deflated, dimpled, or crumpled sphere, and thus tends tohave a lowered sphericity (e.g., below that of 0.9). Similarly, when asignificant rupture occurs, the spherical particles may take on the formof angular shards or fragments which have a substantially differentshape than highly spherical particles. Therefore, uniformity may also beused to measure the mechanical resistance to rupture, with adequateuniformity (e.g., no more 10% of particles having a varied shape) beingan indicator for high mechanical strength of the produced hollow silicaspheres.

In some embodiments, the silica-containing shell has a thickness ofabout 150 to 210 nm, preferably 160 to 200 nm, preferably 170 to 190 nm,preferably 180 to 185 nm. In some embodiments, the core has a diameterof about 100 to 230 nm, preferably 110 to 220 nm, preferably 120 to 210nm, preferably 130 to 200 nm, preferably 140 to 190 nm, preferably 150to 180 nm, preferably 160 to 170 nm. In preferred embodiments, thesilica-containing shell is of “uniform thickness”, meaning an averageshell thickness that differs by no more than 10%, no more than 8%, nomore than 6%, no more than 4%, preferably no more than 2%, preferably nomore than 1% at any given location on the silica-containing shell.

In some embodiments, the methods herein produce hollow silica sphereswith an average diameter of 490 to 540 nm, preferably 500 to 530 nm,preferably 505 to 525, preferably 510 to 520, with the diameter beingthe longest linear distance measured from one point on the particlethough the center of the particle to a point directly across from it.Instead, when no calcination procedure is performed, the non-calcinedsilica spheres have much larger particle sizes, with an average diameterof about 760 nm.

“Dispersity” is a measure of the homogeneity/heterogeneity of sizes ofparticles in a mixture. The coefficient of variation (CV), also known asrelative standard deviation (RSD) is a standardized measure ofdispersion of a probability distribution. It is expressed as apercentage and may be defined as the ratio of the standard deviation (σ)to the mean (μ, or its absolute value |μ|, and it may be used to showthe extent of variability in relation to the mean of a population. In apreferred embodiment, the hollow silica spheres of the presentdisclosure have a narrow size dispersion, i.e., are monodisperse, with acoefficient of variation of less than 30%, preferably less than 25%,preferably less than 20%, preferably less than 15%, preferably less than12%, preferably less than 10%, preferably less than 8%, preferably lessthan 5%, preferably less than 3%, with the coefficient of variationbeing defined in this context as the ratio of the standard deviation tothe mean diameter of the hollow silica spheres.

In some embodiments, the hollow silica spheres produced by the methodsherein are in the form of distinct particles which are not present asagglomerates, meaning the hollow silica spheres are well-separated fromone another and do not form clusters. On the other hand, non-calcinedsilica spheres are typically interconnected forming agglomerates made oftwo or more spheres that share an outer silica boundary.

The methods of the present disclosure advantageously produce hollowsilica spheres having surface characteristics and porosities that makethem suitable for use in a variety of applications, for exampledelivery, adsorption, biosensor, catalysis, and/or cosmeticapplications. Such surface characteristics (e.g., specific surface area,Langmuir surface area, t-pot external surface area, etc.) and porosities(e.g., pore diameters, pore volume, etc.) can be measured, for example,using a gas sorption instrument such as a Micrometrics ASAP 2020 plussystem (Micrometrics, USA). In some embodiments, the hollow silicaspheres are produced with a specific surface area (BET surface area ormultilayer adsorption) in the range of 350 to 450 m²/g, preferably360-440 m²/g, preferably 370-430 m²/g, preferably 380-420 m²/g,preferably 390-415 m²/g, preferably 400-410 m²/g, preferably 405-408m²/g, preferably about 406 m²/g. The specific surface area of the asproduced hollow silica spheres is greater than the specific surface areaof the non-calcined silica spheres, which is about 4-5 m²/g.

In some embodiments, the hollow silica spheres have a Langmuir surfacearea (monolayer adsorption) of 550 to 700 m²/g, preferably 560-690 m²/g,preferably 570-680 m²/g, preferably 580-670 m²/g, preferably 590-660m²/g, preferably 600-650 m²/g, preferably 610-640 m²/g, preferably about635 m²/g. The Langmuir surface area of the hollow silica spheresproduced with the inventive methods is therefore greater than theLangmuir surface area of the non-calcined silica spheres, which is about7.5-8.5 m²/g.

The t-plot method is a well-known technique which allows determining theexternal micro- and/or mesoporous volumes and the specific surface areaof a sample by comparison with a reference adsorption isotherm of anonporous material having the same surface chemistry. In someembodiments, the hollow silica spheres have a t-plot external surfacearea of 40 to 75 m²/g, preferably 45 to 70 m²/g, preferably 50 to 65m²/g, preferably 55 to 60 m²/g, preferably about 58 m²/g. On the otherhand, non-calcined silica spheres have a t-plot external surface area of5-6 m²/g.

In preferred embodiments, the hollow silica spheres of the presentdisclosure have an average pore diameter of 1.7 to 8 nm, preferably 2.0to 6 nm, preferably 2.1 to 4 nm, preferably about 2.2 nm, and a BJHadsorption cumulative pore volume (of pores between 1.7 nm and 300 nm)of 0.02 to 0.035 cm³/g, preferably 0.024 to 0.030 cm³/g, preferably0.026 to 0.028 cm³/g, or about 0.027 cm³/g. In contrast, the averagepore diameter and the BJH adsorption cumulative pore volume (of poresbetween 1.7 nm and 300 nm) for silica spheres which have not beencalcined are 10-11 nm and 0.016-0.017 cm³/g, respectively.

The methods disclosed herein also form robust hollow silica sphereshaving desirable mechanical strength that resist rupture when placedunder certain stresses. One way to test the mechanical strength is tosubject the hollow silica spheres to ultrasonication for 5-10 min at afrequency of 5-30 kHz, preferably 10-25 kHz, preferably 15-20 kHz, andwith a power intensity of 25-50 W/cm², preferably 30-45 W/cm²,preferably 35-40 W/cm² at 20-25° C. A comparison between the number ofbroken/ruptured hollow silica spheres before and after the sonicationusing visual inspection, for example with SEM or TEM images, thenprovides a measure of mechanical strength, in terms of the percentremaining highly spherical (e.g., having an average sphericity of atleast 0.9). In some embodiments, the hollow silica spheres produced bythe methods of the present disclosure remain uniform after subjecting toultrasonication. That is, no more than 10%, preferably no more than 5%,preferably no more than 1% of the distribution of the hollow silicaspheres rupture (have a sphericity of less than 0.9) upon prolongedexposure to ultrasonication. This contrasts to most hollow silicaspheres produced by template methods, which tend to crater or ruptureeasily under mechanical stress and thus have the tendency to benon-uniform, i.e., greater than 10% of a population having a differentshape (Liu et al. “Preparation of hollow silica spheres with differentmesostructures” Journal of Non-Crystalline Solids, 2008, 354, 826-830;Gorsd et al. “Synthesis and Characterization of Hollow Silica Spheres”,Procedia Materials Science, 2015, 8, 567-576).

The methods disclosed herein also advantageously produce hollow silicaspheres which have at least marginal solubility in water and thus can beused more readily in aqueous-based applications, such as in vivo drugdelivery. In some embodiments, the hollow silica spheres have asolubility in water at ambient conditions of 0.1 to 50 mg per 10 mL ofwater, preferably 0.2 to 45 mg per 10 mL of water, preferably 0.5 to 40mg per 10 mL of water, preferably 1 to 35 mg per 10 mL of water,preferably 2 to 30 mg per 10 mL of water, preferably 3 to 25 mg per 10mL of water, preferably 5 to 20 mg per 10 mL of water, preferably 10 to15 mg per 10 mL of water. Conversely, silica spheres which have not beencalcined using the procedures described herein, have limited or nosolubility in water under ambient conditions with solubilities less than0.1 mg per 10 mL of water, preferably less than 0.01 mg per 10 mL ofwater, preferably less than 0.001 mg per 10 mL of water, preferably 0 mgper 10 mL of water. Such low or no aqueous solubility may prohibit theuse of non-calcined silica spheres in certain applications such as drugdelivery without performing surface modification steps to aid theaqueous solubility, which of course comes at the expense of time,scalability, material throughput, and production cost.

While phenyl and other aryl groups may be removed by single or multistepcalcining, the use of particular multi-staged calcining steps provides abenefit. From the above description it is clear that the methods, whichmost notably involve use of a hydrolyzable aryl silane and a multi-stagecalcining procedure, provide hollow silica spheres having superioruniformity, degrees of hollowness, mechanical properties, aqueoussolubility, surface characteristics, etc. compared to non-calcinedvariants. Further, the inventors have unexpectedly discovered thatparticular multi-stage calcining procedures surprisingly provide hollowsilica spheres with superior sphericity, degree of hollowness,uniformity, and/or monodispersity, compared to otherwise identicalprocesses using different calcining programs, for example methodsemploying single-stage calcining procedures. By way of example, when asingle-stage calcination procedure (that is, one that involves rampingfrom one temperature to another at a particular rate without anyintermediate holding steps) is employed that involves calcining theprecipitate by heating up to 600° C. at a ramp rate of 10° C./min, theresulting product has a low sphericity (e.g., less than 0.9), is notuniform (e.g. more than 10% of the distribution have a different shape),is not substantially hollow (e.g., has a degree of hollowness of lessthan 2), and has a low monodispersity (has a particle size coefficientof variation of greater than 30%).

This is surprising since the ramping rate and final calciningtemperature of the single-stage calcination program are similar to thoseemployed in the multi-stage calcining program of the present disclosure.Without being bound by theory, the superior and unexpected resultsdemonstrated may be because the multi-stage (e.g., two-stage)calcination program provides sufficient time for the aryl groups of thehydrolyzable aryl silane to sequester and orient themselves within thecenter of the spherical particles, while the silanol functionalityaggregate to face the surroundings, akin to the packing behavior ofoil-in-water micelles. Therefore, the step-wise temperature increase ofthe multi-stage program may advantageously allow for reorientation ofthe hydrophobic and hydrophilic groups while the aryl groups areultimately being removed through the increasing temperature, therebyforming the hollow core and providing the hollow silica spheres with theaforementioned properties without the need for templates.

The above described advantages enable the hollow silica spheres to beuseful in many applications, including drug delivery/carrierapplications, biosensors, catalysis, cosmetics, adsorbent applications,fillers in polymer, building, or construction applications, and thelike.

In particular, the aqueous solubility properties of the hollow silicaspheres allows them to be used directly as a carrier for sustainedrelease of antitumor agents. For example, the hollow silica spheres maybe loaded with one or more antitumor agents such as adriamycin, taxol,docetaxel, vincristine sulfate, fluorouracil, methotrexatum, novantrone,cyclic adenosine monophosphate, cyclophosphamide, peplomycin sulfate,nitrocaphane, solazigune, aclarubicin hydrochloride, carmustine,temozolomide, lomustine, carmofur, tegafur, dactinomycin, mitomycin,amsacrine, amifostine, cisplatin, alarelin, aminoglute-thimide,chlormethine hydrochloride, and the like, including derivatives thereof,for combating various types of cancers, including, but not limited to,lung cancer, breast cancer, melanoma, colon cancer, pancreatic cancer,glioma, hepatic tumors, pulmonary tumors, bone tumors, adrenal tumorsand other solid tumors. The mode of delivery is not limited and mayinvolve targeted or non-targeted delivery, for example throughcombination with a targeting agent such as tumor specific folic acidligand or a tumor specific antibody.

Further, due to the sphericity, degree of hollowness, high surfaceareas, and porosity, the products formed from the methods disclosedherein are result in small pressure drops, making them especiallysuitable for adsorptive applications for processing of gases, vapors,liquids and solutions. Accordingly, the hollow silica spheres are usefulfor various chromatographic applications.

Substrates for HSS. The hydrolyzable aryl silane employed may be anysilane having at least one aryl substituent and at least onehydrolyzable group bonded directly to the Si atom. Hydrolyzable groupsinclude, but are not limited to, alkoxy groups (e.g., methoxy, ethoxy,propoxy, iso-propoxy, t-butoxy, as well as substituted variants, as wellas mixtures of one or more of these groups) and halo groups (e.g.,chloro, bromo, iodo, and fluoro), including mixtures of alkoxy and halogroups. The hydrolyzable aryl silane may therefore have one, two, orthree hydrolyzable groups, preferably three hydrolyzable groups whichmay be the same or different, most preferably the same.

Likewise, the hydrolyzable aryl silane employed may have one, two, orthree aryl groups, preferably one aryl group. In cases where thehydrolyzable aryl silane contains one aryl group, the hydrolyzable arylsilane may optionally include one or two alkyl or vinyl substituentsbonded directly to the Si atom. The term “aryl”, as used herein, andunless otherwise specified, refers to an aromatic group containingcarbon in the aromatic ring(s), such as phenyl, biphenyl, naphthyl,anthracenyl, and the like, as well as optionally substituted analogsthereof. The term aryl is also meant to include “heteroaryl” groups, oraryl substituents where at least one carbon atom is replaced with aheteroatom (e.g. nitrogen, oxygen, sulfur) so long as the heteroatom isnon-nucleophilic so as to prevent reaction with the hydrolyzable groupof a neighboring hydrolyzable aryl silane. Such heteroaryl groups mayinclude, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl,quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl,oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl,pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thia-diazolyl,isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl,benzodioxolanyl, benzodioxane, and the like, as well as optionallysubstituted analogs thereof. The nitrogen and sulfur heteroatoms mayoptionally be oxidized (i.e., N→O and S(O)p, wherein p is 0, 1 or 2) oroptionally protected with protecting groups as necessary as known tothose skilled in the art, for example, as taught in Greene, et al.,“Protective Groups in Organic Synthesis”, John Wiley and Sons, SecondEdition, 1991, hereby incorporated by reference in its entirety. Theterm “substituted” refers to at least one hydrogen atom that is replacedwith a non-hydrogen group, provided that normal valencies are maintainedand that the substitution results in a stable compound. When a compoundis noted as “optionally substituted”, the substituents are selected fromthe exemplary group including, but not limited to, alkyl, cycloalkyl,cycloalkylalkyl, arylalkyl, heteroaryl, aryl, heterocyclyl, alkoxy,cycloalkyloxy, aryloxy, arylalkyloxy, aroyl, alkanoyl, alkanoyloxy,carboxy, alkoxycarbonyl, halo (e.g. chlorine, bromine, fluorine oriodine), dialkylamino, diarylamino, arylalkylamino, alkanoylamino,nitro, cyano, carbamyl, alkylthio, arylthio, arylalkylthio,alkylsulfonyl (i.e. —SO₂alkyl), arylsulfonyl (i.e. —SO₂aryl),arylalkylsulfonyl (i.e. —SO₂arylalkyl), haloalkyl, oxo, and the like.

Exemplary hydrolyzable aryl silanes include, but are not limited to,ethoxy(diphenyl)vinyl silane, trichloro[4-(chloromethyl)phenyl] silane,dimethoxy(diphenyl) silane, diethoxy(diphenyl) silane,diethoxy(methyl)phenyl silane, trichloro(phenyl) silane,triethoxy(phenyl) silane, and trimethoxy(phenyl) silane.

In preferred embodiments, the hydrolyzable aryl silane is atrialkoxy(aryl) silane, more preferably a trialkoxy(phenyl) silane, mostpreferably trimethoxy(phenyl) silane.

It is also envisioned that hydrolyzable arylalkyl silanes may be used inaddition to, or in lieu of the hydrolyzable aryl silane, whereby thearyl group is present but is bonded to the Si atom through an alkylenelinking group. For example, trimethoxy(2-phenylethyl) silane may beused.

In preferred embodiments, the hydrolyzable aryl silane is the onlysource, reagent, or starting material used in the present disclosure tosynthesize the hollow silica spheres that contains aryl functionality.In preferred embodiments, the hydrolyzable aryl silane is the only Sisource utilized in the present method, and other sources of Si, forexample tetraethyl orthosilicate (TEOS), may be optionally excluded.

Hydrolysis.

Hydrolysis may be carried out by dissolving the hydrolyzable aryl silanein the aqueous solution comprising, consisting essentially of, orconsisting of water and an acid with optional stirring and/or heating,for example, heating to a temperature of 30-100° C., preferably 40-90°C., preferably 50-80° C., preferably 55-65° C., preferably 60° C. Theamount of the hydrolyzable aryl silane dissolved in the aqueous solutionmay be varied, although typically a volume ratio of the hydrolyzablearyl silane to the volume of the aqueous solution ranges from 1:50 to1:100, preferably 1:60 to 1:95, preferably 1:70 to 1:90, preferably 1:75to 1:85. The water may be tap water, distilled water, twice distilledwater, deionized water, deionized distilled water, reverse osmosiswater, or various other water sources.

The acid employed in the hydrolysis reaction is preferably a mineralacid such as hydrochloric acid, nitric acid, phosphoric acid, sulfuricacid, hydrobromic acid, perchloric acid, and hydroiodic acid. Inpreferred embodiments, the acid is nitric acid. A concentration of theacid in the aqueous solution may vary widely, but typical concentrationsrange from 1-15 mM, preferably 2-13 mM, preferably 3-11 mM, preferably4-10 mM, preferably 5-9 mM, preferably 6-8 mM.

After combining the hydrolyzable aryl silane with the aqueous solution,the hydrolysis reaction is allowed to take place for an appropriate timeto convert the hydrolyzable aryl silane into a partially or fullyhydrolyzed form, whereby the hydrolyzable group (e.g., methoxy, chloro,etc.) is replaced by —OH, to form a hydrolyzed silane solution. In mostcases, especially when heating is employed, less than 10 minutes,preferably less than 5 minutes, more preferably less than 3 minutes isenough to result in complete hydrolysis, although longer hydrolysistimes may also be employed.

In some embodiments, metal oxides or metal salts, such as Fe₃O₄ orγ-Fe₂O₃ may be incorporated into a mixture to be hydrolyzed, forexample, to form Fe—HSS. Nickel and cobalt are also magnetic materialsbut are toxic. Magnetic cobalt or nickel nanoparticles may beincorporated into HSS by methods similar to those described for Fe₃O₄herein using Co and Ni substrates similar to Fe₃O₄, for example, byadmixture of their oxides (e.g., Co₃O₄ or NiO) or salts with HSSsubstrates and subsequent hydrolyzation and precipitation. The resultingCo—HSS or Ni—HSS may be used in amounts that minimize toxicity or forapplications in which toxicity is not a concern.

Once hydrolysis is deemed sufficiently complete, the hydrolyzed silanesolution may be mixed with an appropriate hydroxide base to condense thehydrolyzed silane thereby forming a precipitate.

Hydroxide base. The hydroxide base employed in the condensation reactionmay be an alkali metal hydroxide (e.g., lithium hydroxide, sodiumhydroxide, potassium hydroxide, rubidium hydroxide, and cesiumhydroxide), an alkali earth metal hydroxide (e.g., magnesium hydroxide,calcium hydroxide, strontium hydroxide, and barium hydroxide), or anammonium hydroxide (e.g., ammonium hydroxide, tetramethylammoniumhydroxide, triethylammonium hydroxide, trimethylanilinium hydroxide,etc.). In preferred embodiments, the hydroxide base is ammoniumhydroxide.

The hydroxide base may be used in the form of a solid such as a powder,beads or pellets, or may be used in the form of an aqueous basesolution. When used as a solid, the hydroxide base is preferably in theform of beads or pellets, more preferably in the form of beads, andstill more preferably in the form of beads having an average beaddiameter of about 0.1 to 2 mm, preferably about 0.2 to 1.5 mm, morepreferably about 0.5 to 1 mm. When the hydroxide base is used in theform of an aqueous base solution, the concentration thereof ispreferably about 10 to 50%, preferably about 20 to 40%, more preferablyabout 30 to 35%, most preferably about 33%, by weight of hydroxide baseper total volume of the aqueous base solution.

In some embodiments, an excess of hydroxide base is combined with thehydrolyzed silane solution. For example, a molar ratio of hydroxide baseemployed in the condensation reaction to the acid employed in thehydrolysis reaction may be about 100:1 to 1000:1, preferably 200:1 to900:1, preferably 300:1 to 800:1, preferably 400:1 to 700:1, preferablyabout 500:1. Upon addition of the hydroxide base, a precipitategenerally forms immediately at ambient temperatures (i.e., 20-25° C.),or alternatively upon optional heating to 30-80° C., or 40-70° C., or50-60° C. The resulting suspension may be allowed to settle, oralternatively may be stirred, for example with a mechanical or magneticstirrer.

Washing Precipitates.

The precipitate may then be separated from the suspension, for exampleby filtration, centrifugation, decantation, and the like, and optionallywashed with an organic solvent, water, or both. Exemplary organicsolvents may include, but are not limited to C₁ to C₄ lower alkanols,for example, methanol, ethanol, isopropanol, butanol; polyols and polyolethers, for example, glycol, 1,3-propanediol, 1,3-butanediol,2-butoxyethanol, propylene glycol, diethylene glycol, ethylene glycolmonomethyl ether, and propylene glycol monomethyl ether. Afterwards, theprecipitate may then be dried at a temperature of 20-150° C., preferably50-120° C., preferably 60-100° C., preferably 80-90° C. under standardpressure or under vacuum. For examples of similarhydrolysis/condensation procedures, see B. P. M. Marini, F. Pilati, andP. Fabbri, Colloids Surf., 2008, A 317, (1-3); Y. Taniguchi, K. Shirai,H. Saitoh, T. Yamauchi and N. Tsubokawa, Polymer, 2005, 46,2541-2547—each incorporated herein by reference in its entirety.

Calcination.

Typically calcination is performed at a temperature that removes fromthe surface of HSS at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,95, 99 or 100% of phenyl or other aryl groups from HSS produced by themethods described herein. Calcination may be used to tune thepharmacological properties of an HSS by selectively removing some or allof the phenyl or other aryl groups on the surface of an HSS.

In some embodiments, the calcining step removes most or preferably allaryl groups that originate from the hydrolyzable aryl silane, from thehollow silica spheres. FTIR can be used to determine thepresence/absence of such aryl groups in the hollow silica spheres. Thepresence of aryl C—H asymmetrical stretching vibration peak at about3100 cm⁻¹ indicates the presence of aryl groups prior to calcination,while the absence of this peak after the calcining procedures describedherein indicates removal of at least 90%, preferably at least 95%,preferably at least 99% by weight of the aryl groups.

The method may next involve calcining the precipitate using amulti-stage calcining procedure, preferably a two-stage calciningprocedure, to form the hollow silica spheres with advantageousproperties as will be discussed hereinafter. In some embodiments, thecalcination step is performed in a furnace using, for example, a pre-settemperature program discussed below, or using other variable temperaturesystems known by those of ordinary skill in the art.

In a first stage of the calcining process, the precipitate may be heatedto a first temperature of 180 to 240° C., preferably 185 to 230° C.,preferably 190 to 220° C., preferably 195 to 210° C., preferably about200° C., with a first ramp rate of 3 to 10° C./min, preferably 3.4 to 9°C./min, preferably 3.6 to 8° C./min, preferably 3.8 to 7° C./min,preferably 4 to 6° C./min, most preferably about 5° C./min. Once thefirst temperature is reached, the first temperature may be held for 2minutes to 2 hours, preferably from 10 minutes to 1.5 hours, preferablyfrom 12 minutes to 1 hour, preferably from 14 to 55 minutes, preferablyfrom 15 to 45 minutes, preferably from 20 to 40 minutes, preferably from25 to 35 minutes, preferably about 30 minutes.

After holding the first temperature, the second stage of the calciningprocess may involve heating to a second temperature of 600 to 740° C.,preferably 610 to 730° C., preferably 620 to 720° C., preferably 630 to700° C., preferably 640 to 680° C., preferably 650 to 670° C.,preferably about 660° C., with a second ramp rate of 0.1 to 4° C./min,preferably 0.3 to 3.5° C./min, preferably 0.5 to 3° C./min, preferably0.8 to 2.5° C./min, preferably 1 to 2° C./min, most preferably about1.5° C./min. Once the second temperature is reached, the secondtemperature may be held for 2 to 24 hours, preferably 4 to 23 hours,preferably 6 to 22 hours, preferably 8 to 21 hours, preferably 12 to 20hours, preferably 14 to 18 hours, preferably 15 to 17 hours, mostpreferably about 16 hours to form the hollow silica spheres of thepresent disclosure.

In some embodiments, other stages may be incorporated into themulti-stage calcining program. For example, a third stage may be addedin between the first and the second stage that holds on a thirdtemperature which is between the first and second temperatures (i.e., anintermediate stage). Likewise, a fourth stage may be added after thesecond stage to hold at a fourth temperature that is higher than that ofthe second temperature to finish the calcining program (i.e., afinishing stage). Various other stages may also be included, as well asother variations known for calcination processes, such as changes ofgaseous atmosphere may be practiced.

Ramping.

In regard to calcination temperatures and procedures, the term “ramp” or“ramping” refers to a nonisothermal state where the temperature isvaried in a particular direction (e.g., increased or decreased) overtime, the purpose of which is to move from one temperature setting toanother. On the other hand, the terms “held” or “holding” herein referto an isothermal state where the referenced temperature (e.g., the firsttemperature or the second temperature) is maintained at a constant ornear constant value (i.e., plus or minus 5° C., preferably plus or minus4° C., preferably plus or minus 3° C., preferably plus or minus 2° C.,preferably plus or minus 1° C.) for a certain period of time. Forexample, when the first temperature is selected to be 200° C., holdingthis first temperature for 25 to 35 minutes means that the temperatureis maintained at 200° C. plus/minus 5° C. for a 25 to 35 minute timeperiod before the temperature is subsequently changed. Therefore, theterms “held” or “holding” distinguish from nonisothermal states (i.e.,during periods of temperature ramping) where the temperature is beingraised or lowered at a particular ramping rate range. Again using theabove example, when the temperature is being ramped from 150° C. to atarget temperature of 250° C. over a certain time period, this scenariowould not constitute a “hold” in temperature even though 200° C. may betransiently achieved in moving from 150° C. up to 250° C.

As will become clear, the methods disclosed herein provide hollow silicaspheres having unexpected and superior monodispersity, uniformity,degree of hollowness, mechanical properties, aqueous solubility, andsurface characteristics compared to those produced without calcinationand those produced using a single-stage calcination program. Inalternative embodiments, other calcining protocols may be used to removephenyl or other aryl groups from the surface of a HSS and thus increasetheir antineoplastic properties.

Further, the methods described herein do not require the use of atemplate for forming the hollow spherical particles, and may thus beconsidered “template-free” method thus it is not required to employ andsubsequently dispose of a sacrificial core used by other methods formaking HSS.

The synthesized hollow silica spheres may be used “as is”, or may befurther functionalized to suit a particular application, for example,for use in slow release or pH-responsive drug delivery applications orother carrier applications, biosensors, catalysis, cosmetics, adsorbentapplications, fillers in polymer, building, or constructionapplications, etc. Indeed, the hollow silica spheres may be surfacemodified by coating/grafting with poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA), bi-reactive silanes such as gyycidyl-contianingsilanes, e.g., (3-glycidyloxypropyl) trimethoxysilane (GTPMS), cationicpolysaccharides such as chitosan or various other coatings known bythose or ordinary skill in the art.

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal; e.g., human,non-human primate, cow, pig, camel, llama, horse, goat, rabbit, sheep,hamsters, guinea pig, cat, dog, rat, and mouse. The subject may be ahuman or a non-human. The subject or patient may have undergone or beundergoing other forms of treatment, for example surgical reduction of aneoplasm, radiological or chemotherapeutic treatment. A subject may bemale or female, young or old, for example, <1, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100or >100 years old or any intermediate value within this range.

In typically embodiments, the subject will have a neoplasm, such ascancer, precancer, or a benign neoplasm. A neoplasm is a type ofabnormal and excessive tissue growth called neoplasia. The growth of aneoplasm is typically uncoordinated with that of the normal surroundingtissue and it persists growing abnormally, even if the original triggeris removed. This abnormal growth usually but not always forms a masswhich is typically called a tumor. ICD-10 classifies neoplasms into fourmain groups: benign neoplasms, in situ neoplasms, malignant neoplasms,and neoplasms of uncertain or unknown behavior Malignant neoplasms arealso simply known as cancers and are the focus of oncology. Examples ofsuch subjects include those with colon or colorectal cancer,non-melanoma skin cancer, breast cancer, lung cancer, prostate cancer,melanoma, bladder cancer, non-Hodgkin's lymphoma, kidney cancer,leukemia, pancreatic cancer, thyroid cancer, liver cancer, endometrialcancer, throat cancer, ovarian cancer, or testicular cancer.

Subjects having benign neoplasms or pre-cancerous tumors may also beselected for treatment. Benign neoplasms include skin moles, skin tags(acrochordons), cysts in sebaceous glands (sweat glands), breast cysts,encapsulated skin growths such as those triggered by an insect bite orinfection, raised scar tissue including keloids, and uterine fibroids.

Modes of Administration.

The terms “administration” or “administering” as used herein describes aprocess by which the disclosed HSS compositions can be delivered to asubject. Administration will often depend upon the amount of compositionadministered, the number of doses, and duration of treatment. Multipledoses of the composition may be administered. The frequency and durationof administration of the composition can vary, depending on any of avariety of factors, including patient response. The exosome compositionsmay be administered to the subject by any suitable route. For example,the compositions may be administered parenterally, e.g., by intravenous,subcutaneous, topical, transdermal, intradermal, transmucosal,intraperitoneal, intramuscular, intracapsular, intraorbital,intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, or epidural injection, byinfusion, by electroporation, or by co-administration as a component ofany medical device or object to be inserted (temporarily or permanently)into a subject. For example, the exosome compositions may beadministered intranasally.

The HSS of the invention may be administered by any route that bringsthem into contact with target neoplastic cells. HSS may be administeredsystemically, for example, intravenously, or regionally, for example,into an artery that leads to body location containing a tumor.Typically, the HSS are administered parenterally or in in situ to thesite of a tumor or cancer cells, though other modes of administrationmay be selected depending on the type and location of the neoplasm. Insome embodiments of the invention HSS are injected directly intoneoplasm or tissue or organ containing neoplastic cells.

A “therapeutically effective amount,” or “effective dosage” or“effective amount” as used interchangeably herein unless otherwisedefined, means a dosage of HSS or other drug or active ingredienteffective for periods of time necessary, to achieve the desiredtherapeutic result such regression of a tumor or other neoplasm. Asuitable single dose size is a dose that is capable of inducing orsustaining regression or destruction of a neoplasm in a subject whenadministered one or more times over a suitable time period. An effectivedosage may be determined by a person skilled in the art and may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the drug to elicit a desired responsein the individual. Therapeutically effective amounts for the disclosedHSS compositions can be readily determined by those of ordinary skill inthe art. A therapeutically effective amount may be administered in oneor more administrations. A HSS composition may be given as apreventative treatment or therapeutically at any stage of neoplasticgrowth. The applications and dosages for an HSS composition are notlimited to a particular formulation, combination or administrationroute. The times of administration and dosages used will depend onseveral factors, such as the neoplastic disease state, age, sex, andweight of the individual, and the ability of the composition to elicit adesired response in the individual. Administration may be adjustedaccording to individual need and professional judgment of a personadministrating or supervising the administration of the HSS compositionsof the present invention.

Any dosage of the HSS as disclosed herein that is effective to inhibitneoplastic growth or induce cytotoxicity may be used. A dosagecontaining <0.01, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1,000, 2,000, 3,000 or >3,000 μg of HSS per kilogram of body weight of asubject can be used. This range includes all intermediate values andsubranges. Dosages may be modified based on mode of administration, forexample, a larger dosage may be administered intravenously than a dosageadministered in situ to a neoplasm or to a dosage administeredtopically.

Administration of HSS or HSS loaded with other active ingredients may beas a single dose or multiple doses over a period of time. An HSScomposition may be administered to the patient at any frequencynecessary to achieve the desired therapeutic effect. For example, it maybe administered continuously, once to several times every month, everytwo weeks, every week, or every day. Administration of an HSScomposition may be repeated until the desired therapeutic effect hasbeen achieved. For example, an HSS composition may be administered onceto several times over the course of 1 day, 3 days, 5 days, 1, 2, or 3weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or more than 12months. In some embodiments, compositions containing HSS may beadministered before surgery to shrink a tumor or after surgery as anadjuvant chemo therapy.

An amount of HSS in a therapeutic composition to be administered maydepend on a variety of factors, such as the route of administration andthe seriousness of the condition, and should be decided according to thejudgment of the practitioner and each patient's circumstances. An HSScomposition may be administered to a subject in any amount suitable forthe prevention or treatment of a neoplasm, including cancers or tumors.An effective amount of an HSS composition may partial or completenecrosis or apoptosis of cancer cells, a reduction in tumor size orcancer load, increased cytotoxicity, or a reduction in cancer cellgrowth rate.

Suitable dosage ranges for the kinds of HSS disclosed herein includefrom about 0.001 μg HSS/kg body weight to about 100 mg/kg, about 0.01μg/kg to about 90 mg/kg, about 0.1 μg/kg to about 80 mg/kg, about 1μg/kg about 70 mg/kg, about 10 g/kg to about 60 mg/kg, about 0.1 mg/kgto about 50 mg/kg, about 0.5 mg/kg to about 25 mg/kg, about 1 mg/kg toabout 10 mg/kg, or about 2.5 mg/kg to about 5 mg/kg. For example,suitable dosage ranges of HSS include about 0.001 μg/kg, about 0.01μg/kg, about 0.1 μg/k, about 1 μg/kg, about 10 μg/kg, about 0.1 mg/kg,about 0.5 mg/kg, about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80mg/kg, about 90 mg/kg, or about 100 mg/kg. In vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

Combination Therapy.

Anticancer agents that may be administered using HSS or may becoadministered with HSS include 5-flurouracil, capcitbaine, irinotecan,oxaliplatin, trifluridine and tipiracil (Lonsurf) or combinationsthereof which are often used to treat colorectal cancer. In acombination therapy the dosage of one or more of the active agents maybe reduced thus reducing side-effects associated with a larger dosage.For example, side-effects such as hair loss, mouth sores, loss ofappetite, nausea and vomiting, increased risk of infection, easybruising, fatigue, hand-foot syndrome, neuropathy, allergic orsensitivity reactions, and diarrhea associated with therapy with one ormore conventional drugs used to treat colorectal cancer or other kindsof cancer may be reduced by co-administration of HSS with lower dosagesof the conventional drugs such as those named above.

HSS Conjugates or Platforms.

HSS may incorporate or be coated with targeting moieties such asantibodies or other that bind to cancer cell antigens. Targetingantibodies or antibody fragments containing an antigen binding siteinclude those that bind to CEA which is associated for example withcolon cancer or those that bind to alpha fetoprotein (e.g., liver andtesticular cancer), CA15.3 (e.g., breast cancer), CA 19.9 (e.g.,gastric/pancreatic cancer), CA125 (e.g., reproductive system cancers),or EVP (e.g., nasopharyngeal cancer).

Fe—HSS may be used in applications where magnetic responsiveness ordetection is required, such as the magnetic localization of administeredFe—HSS or drug-loaded Fe—HSS to a particular part of the body. Fe—HSSmay also be used in a variety of bioimaging applications.

Other applications of the HSS disclosed herein include their use asnanoparticle collectors, catalysis, and the adsorption and separation ofgas and pollutants. For example, they may be incorporated into a systemfor adsorption or separation of a liquid or gaseous mixture, as aplatform for a catalyst, as thermal or electrical insulators, as amembrane component, as a component of a superhydrophobic surface.

Having generally described this disclosure, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified. The examples below are intended tofurther illustrate protocols for preparing and testing the hollow silicaspheres and they are not intended to limit the scope of the claims.

Examples

Materials.

Phenyltrimethoxysilane (PTMS), ammonium hydroxide (NH₄OH), and nitricacid (HNO₃) were obtained from Sigma-Aldrich, Inc. Iron oxide (Fe₃O₄)nano-powder was supplied from US Research Nanomaterials, Inc. Ultrapurewater was produced using a Milli-Q water purification system (Bedford,USA).

The synthesized products were labelled as, uncalcined hollow silicaspheres (u-HSS), calcined hollow silica spheres (c-HSS) and iron oxidenanoparticles (Fe₃O₄) incorporated hollow silica spheres (Fe—HSS). Thescheme of the preparation reaction of Fe—HSS is shown in FIG. 1.

Preparation of Uncalcined Hollow Silica Spheres (u-HSS).

The hollow silica spheres (HSS) were synthesized substantially asdescribed by Akhtar et al., J. Saudi Chem. Soc., A novel approach toproduce monodisperse hollow pure silica spheres (available online Sep.22, 2018; incorporated by reference).

Briefly, PTMS was dissolved in HNO₃ and stirred in an isothermal waterbath at 60° C. A solution of NH₄OH swas then added which triggered acondensation reaction. Correspondingly, at the adjacency of thePTMS/water interface, the hydrolysis reaction progressed quickly within3 minutes of the initial acidic condition. The clear mixture rapidlytransformed to white colour solution. Precipitated particles wereremoved from the condensed solution by centrifugation and then washedwith ethanol and subsequently with water. The product was dried at 70°C. and labelled as u-HSS.

Preparation of Calcined Hollow Silica Spheres (c-HSS).

Approximately 100 mg of the synthesized u-HSS were taken and kept inafurnace at the temperature 200° C. for 30 min. Calcination of the u-HSswas performed at 660° C. for 16 hrs. The first and second temperatureramping rates were 5 and 1.5° C./min, respectively. The calcined productwas labelled as c-HSS and is shown in FIG. 2A.

Preparation of Magnetic Nanoparticles Doped HSS (Fe—HSS).

Magnetic nanoparticle-doped HSS were produced by introducing 60 mg ofiron oxide (Fe₃O₄) powder into 50 ml of 6.6 mM HNO₃ solution in a 3-neckround bottom flask which was shaken for 10 minutes.

Then 0.6 ml PTMS was added into the solution and the mixture was againshaken for 10 minutes.

The resulting mixture was then placed into water bath at 60° C. Then 8.5ml of a NH₄OH solution (33%) was added producing a milky solution whichwas kept in the water bath for 1 hour. The water bath was allowed tocool to room temperature and the mixture was then removed. The finalmixture was stirred for about 16 hours at room temperature.

Precipitated particles were recovered from the condensed solution bycentrifugation. The precipitated particles were initially washed withethanol and then with water and the product was dried at 70° C. andlabelled as Fe—HSS as shown by FIG. 5B.

Characterization of HSS.

The sizes and structures of the Fe₃O₄ nanoparticles were analyzed byTransmission Electron Microscopy (TEM) (Model:FEI, Morgagni 268, CzechRepublic). The morphological features of the u-HSS, c-HSS and Fe—HSSwere also examined by TEM. For that purpose, a droplet of suspension ofeither HSS or Fe—HSS was deposited onto TEM grid supported by carbonsupport film. TEM was operated at accelerating voltage of 80 kV. Severalimages were taken to obtain the size of the nanoparticles and the Gatandigital micrograph software was applied to measure the size of theparticles. More than 250 individual Fe₃O₄-particles were measured toobtain a size histogram. The crystalline structure of the nanoparticleswas verified by selected area electron diffraction (SAED) pattern.Furthermore, the overall morphology of u-HSS, c-HSS and Fe—HSS productswere examined by Scanning Electron Microscopy (SEM) (Model: FEI, InspectS50, Czech Republic) where SEM was operated at 20 kV as shown by FIGS.1D-1F.

X-ray diffraction (XRD) of Fe—HSS specimen was performed to confirm theexistence of Fe₃O₄ in the silica spheres. An XRD pattern was taken by anX-ray Diffractometer obtained from Rigaku, Japan by using Cu-Kαradiations (λ=0.154 nm) within the 2θ range of 15°-70°. The XRDinstrument was operated at 40 kV and 15 mA.

Thermal Stability.

The thermal stability of u-HSS, c-HSS and Fe—HSS were examined byThermogravimetric analysis (TGA) and the analysis was carried out usinga thermal analyzer (STA, Parkin-Elmer) 6000, USA. TGA data of sampleswere obtained between temperatures ranging from 25 to 700° C. with aheating rate of 10° C./min under a nitrogen atmosphere. Nitrogen flowrate was maintained at 20 ml/min.

Treatment of Cancer Cells with c-HSS, u-HSS2, and Fe—HSS.

Human colorectal carcinoma (HCT-116) cells were grown as previouslydescribed by Khan F A, Akhtar S, Almohazey D, Alomari M, Almofty S A,Eliassari A. Fluorescent magnetic submicronic polymer (FMSP)nanoparticles induce cell death in human colorectal carcinoma cells.Artif Cells Nanomed Biotechnol. 2018 Jul. 25:1-7. doi:10.1080/21691401.2018.1491476. In brief, HCT-116 cells were grown inDMEM, supplemented with 10% fetal bovine serum, L-glutamine, seleniumchloride, penicillin, and streptomycin. The cells were seeded in 96 wellplates and grown to 80% confluence in a CO₂ incubator(Thermo-scientific, Waltham, Mass., USA) in an atmosphere containing 5%CO₂ at 37° C. Then, the cells were treated with c-HSS, u-HSS, or Fe—HSSeach at the following concentrations 1 mg/mL, 3 mg/mL, 5 mg/mL, and 7mg/mL respectively. After 48 hours, the cells were microscopicallyobserved. Triplicate samples for each grouping were prepared forstatistical evaluation.

Cancer Cell Morphology.

After the treatments with c-HSS, u-HSS2, and Fe—HSS, the cancer cellswere observed microscope (TS100F-Eclipse, Nikon, Japan) to evaluate theanatomical and morphological changes and each sample was observed under200 and 400 magnifications respectively.

Cancer Cells Viability Determined by MTT Assay.

To examine the effects of exposure of cancer cells to c-HSS, u-HSS, andFe—HSS an MTT assay was performed. The cancer cells were seeded with6×10⁴ cells/mL concentration in 96-well culture plates containing DMEM,supplemented with 10% Fetal bovine serum, penicillin and streptomycinand were incubated in CO₂ incubator till they reached 80% confluence.Then, cancer cells were treated with c-HSS, u-HSS, and Fe—HSS at thefollowing concentrations: 1 mg/mL, 3 mg/mL, 5 mg/mL, and 7 mg/mLrespectively. The c-HSS, u-HSS, and Fe—HSS were not added to the controlgroups.

MTT (5 mg/mL) solution was added and cells were incubated for 4 hrs inthe CO₂ incubator and finally media was changed with the addition ofDMSO. The samples were processed for OD reading using ELISA plate reader(Biotek Instruments, Winooski, USA) at 570 nm wavelength. The percentage(%) of cell viability was calculated as per below given formula:

% of Cell viability=(Optical density (OD) of c-HSS,u-HSS,Fe—HSS-treatedcells)/(Optical density (OD) of control cells×100)

Statistical Analysis.

The mean±standard deviation (SD) from control and c-HSS, u-HSS2, andFe—HSS treated groups were calculated. All statistical analyses werecompleted with GraphPad Prism 6 (GraphPad Software). The differencebetween control and c-HSS, u-HSS, and Fe—HSS groups by a one-wayanalysis of variance (ANOVA) test where (P<0.05) was consideredstatistically significant.

Morphological and Structural Analysis.

SEM and TEM are widely used to analyze the surface morphology andstructure of the nanomaterials at high resolution. FIGS. 2A-2F show themorphological analysis of the Fe₃O₄ particles and hollow silica spheres(HSS) synthesized under different conditions. The nano-powder of Fe₃O₄exhibited the spherical-shaped particles with a bit of agglomeration(FIG. 2A). Several particles, more than 250 individual particles wereselected from different TEM images and measured.

The results of this measurement are drawn in the form of size histogram(FIG. 2B). The average diameter of the particles was found 14.2±1.4 nm.

The Fe₃O₄ nanoparticles showed the polycrystalline nature when analyzedby selected area electron diffraction (SAED) pattern (FIG. 2C). Thefirst 5-rings of the SAED pattern started from the inner ring areindexed as, (220), (311), (400), (511) and (440), confirming thestructure of Fe₃O₄ crystal.

FIGS. 2D-2F show the SEM micrographs of u-HSS, c-HSS and Fe₃O₄ doped HSS(Fe—HSS) specimens.

As shown by the TEM analysis, the silica spheres appeared monodispersedand showed well-organized spherical shape having smooth and uniformtexture. The size of u-HSS was measured which was found to be around 760nm (FIG. 2D).

Upon applying the calcination step, the size of c-HSS was reducedsubstantially (FIG. 2E).

The average diameter of the c-HSS was estimated to be 515±15 nm. Asimilar size was found for the magnetic nanoparticles incorporatedsilica specimen (Fe—HSS), see FIG. 2F.

All prepared products, u-HSS, c-HSS and Fe—HSS were further analyzed byTEM (FIG. 3).

FIG. 3A displayed the morphology of several uncalcined spheres (u-HSSspecimen). As analyzed by TEM, the u-HSS were larger in diametercompared to c-HSS and Fe—HSS specimens (see FIGS. 3A-3C). This wasexpected due to presence of phenyl groups with silica spheres. Thisresult is consistent with the observation made earlier by SEM (see FIG.3D-3F).

Furthermore, the TEM images resolved the hollow structure of the spheresas evident by the bright contrast in the middle of the spheres. Theshells of the spheres are appeared darker compared to hollow cores dueto their solid nature. The shell/core structure is more evident andvisible for the c-HSS specimen (FIG. 3B), where the brightness of theimage is increased a lit bit to clarify the core and shell.

TEM micrographs showed the clear evidence of the core structure of thesilica spheres. It was found that the shells were larger in thicknessthan the size of the cores.

FIGS. 3C and 3D showed the TEM micrographs of Fe—HSS specimen at twomagnifications.

Excitingly, the Fe₃O₄ nanoparticles were seen in the core of the silicaspheres. Few such Fe₃O₄ nanoparticles inside the core are highlighted bywhite arrows in FIG. 3D, identified by their darker contract compared tohollow core.

SEM and TEM analysis confirm the successful loading of Fe₃O₄nanoparticles to hollow silica spheres.

The presence of Fe₃O₄ in the HSS product was verified by performingenergy dispersive X-Ray specroscopy (EDS). The EDS analysis confirms thepresence of iron in the HSS product, where about 5% Fe was found in theFe—HSS specimen.

In addition to SEM and TEM, X-Ray diffraction (XRD) analysis wasperformed on Fe—HSS specimen to confirm the presence of Fe₃O₄nanoparticles in the HSS. FIG. the 4 shows the results of XRD powderpattern of Fe—HSS within the 2θ range of 15°-70°. The broad diffractionpeak at 20° marked with * symbol is ascribed to the amorphous silica.The XRD patterns of u-HSS showed that silica spheres show the similarpattern. The diffraction peaks of [2 2 0], [3 1 1], [4 0 0], [4 4 0],and [5 1 1] proved the existence of Fe₃O₄ spinel structure in the HSS,thus confirming the presence of magnetic nanoparticles within the silicaspheres. This result is in good agreement with electron diffraction datashown in FIG. 2C, where Fe₃O₄ nanoparticles were analyzed by SAEDpattern obtained by TEM. The intense peak/ring in both cases is (311).

The physical apperance of two products, c-HSS and Fe—HSS, is shown byFIGS. 5A and 5B, respectively. The c-HSS specimen showed the white milkycolour whereas Fe—HSS powder gave the blackish appearance, indicatingthe presence of iron oxide in this product.

The presence of Fe₃O₄ nanoparticles in Fe—HSS was also confirmed byperforming a simple test as shown by FIGS. 5C and 5D. The Fe—HSS powderwas pulled by magnetic slab as shown by FIG. 5D, while c-HSS specimenshowed no attraction to the magnetic slab.

The Fe—HSS powder attached to magnetic slab can be seen in FIG. 5D,confirming the presence of magentic nanoparticles in the sample. FIG. 5Cshows the same slab without magnetic particles of Fe—HSS attached to it.

TG Analysis.

Thermo-gravimetric analysis (“TGA”) of c-HSS, u-HSS and Fe—HSS is shownin FIG. 6. TGA plots of the various HSS were prepared between thetemperature ranging from 25° C. to 750° C. A minor weight loss of HSSbetween the temperature ranges from 25 to 400° C. was observed. Theu-HSS exhibited a single step degradation starting with the temperature400° C., which is corresponded to the loss of phenyl groups. Inaddition, we have also observed significant weight loss for u-HSS andmagnetic nanoparticles after 550° C. On the other hand, the c-HSS showsno visible weight change and remain constant during 25° C. to 750° C.The results were further explained by TEM analysis in FIG. 2, where a30% (760 to 510 nm) reduction in the average size of the spheres wasobserved. This reduction in HSS was caused by loss of phenyl groupsafter calcination at the temperature of 660° C. The thermal stability ofthe calcined product is explained in FIG. 6 where the organic-naturedphenyl molecules were removed slowly without changing the morphology ofHSS. By removing the organic groups from the final product, the thermalstability of HSS was obtained.

Effect of u-HSS on Cancer Cell Morphology.

The treatment of u-HSS (uncalcined HSS) with dose 1 mg/mL showed littleimpact on the cancer (FIG. 7B) with compared to control cells (FIG. 7A).The dose of 4 mg/mL showed moderate nuclear condensation andaugmentation of cancer cells (FIG. 7C). Whereas, the dosages of 5 mg/mLand 7 mg/mL showed some changes in cancer cell morphology but not manycell deaths (FIG. 7D).

Effect of c-HSS on Cancer Cell Morphology.

Post 48-hour treatment of c-HSS (calcined HSS) with dose 1 mg/mL showedmoderate levels of nucleus condensation and nuclear augmentation of theHCT-116 cells (FIG. 8B) with compared to control cells (FIG. 8A). Thedose of 3 mg/mL showed strong nuclear condensation and augmentation andshowed the beginning of cell membrane disruption (FIG. 8C). The dosagesof 5 mg/mL and 7 mg/mL showed a significant loss of cell population(FIG. 8D).

Effect of Fe—HSS on Cancer Cell Morphology.

The treatment of cells with Fe—HSS (1 mg/mL) showed strong nucleuscondensation and nuclear augmentation of the cancer cells (FIG. 9B). Nomorphological changes in the control cells (FIG. 9A) were observed. Thedose of 3 mg/mL showed further nucleus condensation and augmentation(FIG. 9C), whereas the dosages of 5 mg/mL and 7 mg/mL showed asignificant loss of cell population (FIG. 9D).

Cancer Cells Survivability.

MTT assays were carried out to examine cell viability and the inhibitionrate of HCT-116 cell line. MTT assay was carried with differentconcentrations of u-HSS, c-HSS2, and Fe—HSS for 48 hrs. The treatmentusing 1 mg/mL, 3 mg/mL, 5 mg/mL, and 7 mg/mL) of u-HSS showed 85%,79.68%, 75.52, and 66.40% cancer cell viability (FIG. 10), whereas thetreatment of c-HSS using 1 mg/mL, 3 mg/mL, 5 mg/mL, and 7 mg/mL showed79.42%, 76.82%, 65.18%, and 34.56% cell viability respectively (FIG.11). When cancer cells were treated with Fe—HSS using 1 mg/mL, 3 mg/mL,5 mg/mL, and 7 mg/mL, the cancer cell viability was significantlyreduced to 53%, 46.48%, 37.63% and 31.26% respectively (FIG. 12).

As shown by the work described above, the inventors have synthesizedu-HSS, c-HSS, and Fe—HSS and all these HSSs were found to be highlysoluble in water. The physical properties of the u-HSS, c-HSS2, andFe—HSS were characterized. The size and structure and morphologicalfeatures of the uncalcined (u-HSS), calcined (c-HSS) and Fe3O4 doped HSS(Fe—HSS) were examined by taking droplet of suspension of either HSS orFe—HSS onto TEM grid with carbon support film. SEM and TEM are widelyused to analyze the surface morphology and structure of thenanomaterials at high resolution. The nano-powder of Fe₃O₄ exhibitedspherical-shaped particles with a bit of agglomeration, severalparticles, more than 250 individual particles were selected fromdifferent TEM images and measured. The average diameter of the particleswas found 14.2±1.4 nm. The Fe3O4 nanoparticles showed thepolycrystalline nature when analyzed by selected area electrondiffraction (SAED) pattern. The first 5-rings of the SAED patternstarted from the inner ring are indexed as, (220), (311), (400), (511)and (440), confirming the structure of Fe₃O₄ crystal as reported byother researchers; Xiong, Z., et al., A facile method for theroom-temperature synthesis of water-soluble magnetic Fe3O4nanoparticles: Combination of in situ synthesis and decomposition ofpolymer hydrogel. Materials Chemistry and Physics, 2011. 130(1-2): p.72-78; Liu et al., 2015. The average size of the u-HSS was measuredaround 760 nm, which was found to be consistent with the size found byanother researcher; Hah, H. J., et al., Simple preparation ofmonodisperse hollow silica particles without using templates. ChemicalCommunications, 2003(14): p. 1712-171.

The XRD patterns of u-HSS and c-HSS were shown in our previous work(Akhtar et al., 2018), where silica spheres showed the similar pattern.The diffraction peaks of [2 2 0], [3 1 1], [4 0 0], [4 4 0], and [5 1 1]proved the existence of Fe₃O₄ spinel structure in the HSS (Liu et al.,2017) which confirmed the presence of magnetic nanoparticles within thesilica spheres. The thermal stability of the calcined product can beexplained by applying a calcination step, where the organic-naturedphenyl molecules were removed slowly without changing much themorphology of HSS. The thermal stability of HSS was obtained by theremoval of these organic groups from the final product.

After confirming the physical and structural properties of u-HSS, c-HSS,and Fe—HSS, their properties as anti-cancer agents were evaluated. Humancolorectal carcinoma cells (HCT-116) were used to evaluate anti-cancerproperties of —HSS, c-HSS, and Fe—HSS. HSS were found to produceconcentration dependent effects on cancer cells. The microscopicexamination of both control and —HSS, c-HSS, and Fe—HSS treated cancercells showed that u-HSS, c-HSS, and Fe—HSS not only induced nuclearcondensation, augmentation and disintegration but also affected thecancer cell membrane. Thus, u-HSS, c-HSS, and Fe—HSS are all highlyeffective in attenuating cancer cells proliferation, however, theresponse of c-HSS (calcined HSS) was better than u-HSS (uncalcined HSS)and Fe—HSS was more effective in reducing cancer cell proliferationcompared to u-HSS and c-HSS. While not being bound to any theory orexplanation, the inventors consider that the absence of phenyl groups inthe c-HSS and Fe—HSS makes them more effective in targeting cancer cellsand inducing cytotoxicity.

As shown by the above Examples, the inventors synthesized differentforms of hollow silica spheres (HSS) with Fe₃O₄ (Fe—HSS) and withoutFe₃O₄ (u-HSS and c-HSS) and tested their anti-cancer aptitudes on thecancer cells. They found that all forms of HSS reduced cancer cellsproliferation with dose dependent manner. The lower dosage (1 mg/mL)produced minor effect on the cancer cells proliferation, while thehigher dosages (3, 5 and 7 mg/mL) significantly reduced the cancer cellproliferation during the same period (48 hrs). In addition, theinventors have shown that u-HSS, c-HSS and Fe—HSS treated cancer cellsundergo nuclear disintegration and fragmentation. The inventors alsoidentified a differential action of u-HSS, c-HSS, and Fe—HSS, forexample, Fe—HSS was more effective in reducing cancer cell proliferationcompared to u-HSS and c-HSS and u-HSS was less effective than c-HSS.These surprising results indicate the usefulness of hollow silicaspheres, particularly those from which phenyl/aryl groups have beenremoved, or those containing Fe₃O₄, are for biological application suchas treatment of neoplasms.

Terminology

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by deletion of http: or by insertion of a space orunderlined space before www. In some instances, the text available viathe link on the “last accessed” date may be incorporated by reference.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included. For example, if a particular elementor component in a composition is said to have 8 wt. %, it is understoodthat this percentage is in relation to a total compositional percentageof 100%, unless stated otherwise.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper”, “in front of” or “behind” and the like, may be used herein forease of description to describe one element or feature's relationship toanother element(s) or feature(s) as illustrated in the figures. It willbe understood that the spatially relative terms are intended toencompass different orientations of the device in use or operation inaddition to the orientation depicted in the figures. For example, if adevice in the figures is inverted, elements described as “under” or“beneath” other elements or features would then be oriented “over” theother elements or features. Thus, the exemplary term “under” canencompass both an orientation of over and under. The device may beotherwise oriented (rotated 90 degrees or at other orientations) and thespatially relative descriptors used herein interpreted accordingly.Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal”and the like are used herein for the purpose of explanation only unlessspecifically indicated otherwise.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

1. A method for treating a subject having a neoplasm, comprising:administering to the subject a composition comprising Fe₃O₄-hollowsilica spheres (“Fe—HSS”), wherein the subject is in need of treatmentfor the neoplasm.
 2. The method of claim 1, wherein the hollow silicaspheres are prepared by a process comprising hydrolysis of a mixturecomprising Fe₃O₄ and phenyl-tri-methoxysilane (“PTMS”) or anotherhydrolyzable aryl silane followed by condensation with a hydroxide base.3. The method of claim 1, wherein the Fe—HSS are not calcined.
 4. Themethod of claim 1, wherein the Fe—HSS are calcined.
 5. The method ofclaim 1, wherein the surfaces of the Fe—HSS are substantially free ofphenyl or aryl groups.
 6. The method of claim 1, wherein the Fe—HSSfurther comprise at least one anticancer drug or agent.
 7. The method ofclaim 1, wherein the neoplasm is cancer.
 8. The method of claim 1,wherein the neoplasm is colon or colorectal cancer.
 9. The method ofclaim 1, wherein the neoplasm is non-melanoma skin cancer, breastcancer, lung cancer, prostate cancer, melanoma, bladder cancer,non-Hodgkin's lymphoma, kidney cancer, leukemia, pancreatic cancer,thyroid cancer, liver cancer, endometrial cancer, throat cancer, ovariancancer, or testicular cancer.
 10. The method of claim 1, wherein theneoplasm is a benign neoplasm or a pre-cancerous tumor.
 11. The methodof claim 1, wherein the composition comprising the HSS is administeredin situ to the site of a tumor or a tissue or organ containing cancercells.
 12. The method of claim 1, wherein the composition comprising theHSS is administered intravenously, intramuscularly, subcutaneously, orotherwise parenterally.
 13. A sterile composition, comprising: hollowsilica spheres that incorporate Fe₃O₄ (“Fe—HSS”) that have not beensintered and a pharmaceutically acceptable excipient or carrier.
 14. Thecomposition of claim 13, wherein the Fe—HSS have average diametersranging from 415 to 615 nm.
 15. The composition of claim 13, wherein theHSS is Fe—HSS that comprises Fe₃O₄ particles having an average diameterranging from 9.5 nm to 21.3 nm.
 16. The composition of claim 13, whereinthe Fe3O4 particles are crystalline and present in an amount rangingfrom 0.1 to 10 wt % of a combined weight of the HSS and Fe₃O₄ in theFe—HSS.
 17. The composition of claim 13, further comprising at least oneanticancer drug or agent.
 18. A method for forming hollow silica spheresincorporating Fe₃O₄ (“Fe—HSS”) comprising: dissolving a hydrolyzablearyl silane in an aqueous solution comprising Fe₃O₄, water and an acidto form a hydrolyzed silane solution, wherein a molar ratio of thehydrolysable aryl silane to the Fe₃O₄ ranges from 100:1 to 1:100; mixingthe hydrolyzed silane solution with a hydroxide base to form aprecipitate; and recovering Fe—HSS from the precipitate.
 19. The methodof claim 18, wherein the hydrolyzable aryl silane is trimethoxy(phenyl)silane and the hydroxide base is NH₄OH.
 20. The method of claim 18,wherein the precipitate is not calcined.